Type I interferons (IFN-α and -β subtypes) produce pleiotropic effects on cells, such as inhibition of virus replication (antiviral effect), inhibition of cell proliferation (anti-tumoral effects), and modulation of immune cell activities (immunoregulatory effects). These multiple effects of interferons (IFNs) are correlated with morphological and biochemical modifications of cells (Revel, 1984, for review).
Interferons exert their activities through species-specific receptors. For type I IFNs, two transmembranal receptor chains have been identified: IFNAR1 (Uze et al, 1990) and IFNAR2-2 (or IFNAR2-c, Domanski et al, 1995). Transduction of the signal generated by IFN-α, β, ω involves protein tyrosine kinases of the Janus kinases (Jak) family and transcription factors of the Stat family (Darnell et al, 1994). Proteins of the Jak-Stat pathways are activated by binding to the intracytoplasmic (IC) domains of the IFNAR1 and IFNAR2 receptor chains. Among the proteins binding to the IFNAR1 IC domain are tyk2 and Stat2 (Abramovich et al, 1994). Stat2 would then recruit Stat1 to form the IFN-induced ISGF3 transcription complex which activates IFN-induced genes (Leung et al, 1995). Transcription complexes containing Stat3 are also induced by IFN-β (Harroch et al, 1994) and an IFN-dependent binding of Stat3 to IFNAR1-IC was observed (Yang et al, 1996). Protein-tyrosine phosphatase PTP1C and D reversibly associate with IFNAR1 upon IFN addition (David et al, 1995a). In addition, two serine/threonine protein kinases, the 48 kDa ERK2 MAP kinase (David et al, 1995b) and the cAMP activated protein kinase A (David et al, 1996) bind to the isolated membrane-proximal 50 residues of IFNAR1-IC. Therefore, the type I IFN receptor IC domains serve as docking sites for multiple proteins which serve to generate and regulate the biological effects of IFNs on cells.
Two-hybrid screening in yeast is a potent method for identifying new proteins which bind to defined polypeptide sequences (Fields and Song, 1989). Briefly, the two-hybrid screen is performed by transfecting yeast cells with (a) a plasmid DNA in which the defined polypeptide (bait) is fused to the DNA-binding domain of the Gal4 transcription factor, and (b) a cDNA library fused to the activation domain of Gal4 in a pACT plasmid. Yeast cells transfected with a cDNA that encodes for a protein which binds to the polypeptide bait will then reconstitute the Gal4 activity. The presence of such a protein which binds the polypeptide bait is revealed by expression of an enzymatic activity, such as β-galactosidase, from a GAL1-lacZ construct that is preferably introduced into the yeast genome. From yeast clones which are positive in this test, it is possible to isolate the pACT plasmid, to determine the nucleotide sequence of its insert and to identify the protein which it encodes. This method has allowed the identification of novel proteins which interact with the IC domain of cytokine receptors (Boldin et al, 1995).
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